目的观察α1抗胰蛋白酶(α1-antitrypsin,AAT)Z型突变体(α1-antitrypsin Z variant,ATZ)表达对细胞自噬水平的影响,探讨ATZ细胞毒作用的可能机制。方法体外培养人胚肾细胞株HEK 293T并转染ATZ真核表达质粒,利用Western blot法检测自噬...目的观察α1抗胰蛋白酶(α1-antitrypsin,AAT)Z型突变体(α1-antitrypsin Z variant,ATZ)表达对细胞自噬水平的影响,探讨ATZ细胞毒作用的可能机制。方法体外培养人胚肾细胞株HEK 293T并转染ATZ真核表达质粒,利用Western blot法检测自噬相关蛋白LC3和p62的表达,免疫荧光染色观察LC3的细胞定位,real-time PCR法检测自噬相关基因Atg5、Atg12和Beclin1的变化,DAB染色观察ATZ过表达对细胞形态的影响。结果与转染空质粒对照组相比,ATZ过表达促进细胞自噬的标志分子LC3由LC3-Ⅰ转变为LC3-Ⅱ,p62水平降低;而经自噬抑制剂氯化铵处理后,ATZ过表达细胞中出现LC3点状聚集,p62水平增加;同时,ATZ过表达明显增加自噬相关基因Atg5、Atg12的mRNA水平,而对Beclin1无明显影响;少量表达ATZ的细胞形态基本正常,细胞中LC3形成点状聚集;反之,过度表达ATZ的细胞核变小、固缩甚至消失,无LC3-Ⅱ表达。结论过表达ATZ通过增加Atg5和Atg12表达激活自噬,这可能与其细胞毒性有一定相关性。展开更多
Autophagy is a highly evolutionarily conserved pathway that depends on lysosome to degrade misfolded proteins and damaged organelles. Besides canonical autophagy, studies have shown some chemicals could bypass sever...Autophagy is a highly evolutionarily conserved pathway that depends on lysosome to degrade misfolded proteins and damaged organelles. Besides canonical autophagy, studies have shown some chemicals could bypass several core genes to induce autophagy, but the targets and regulatory mechanism is still unclear. In this work one novel chemical, G1, was screened out which could trigger both canonical autophagy and non-canonical autophagy by recruiting Atgl6L1 to pre-autophagosomal site and causing LC3 lipidation. The Gl-induced non-canonical auto- phagy was ULK1, and Beclinl-independent but ubiquitin-like conjugation system-dependent, indicating G1 might target the upstream of Atgl6L1. Moreover, inhibition of V-ATPase by specific V-ATPase inhibitiors could suppress the formation of Gl-indueed autophagosomes in FIP200-defieient MEF cells. While other classic lysosomal inhibi- tors could not block the puneta of Atgl2, Atgl6L1 and LC3, in different stages, suggesting V-ATPase activity in- stead of lysosome function is required for Gl-indueed non-canonical autophagy. These studies broaden the under- standing of different working pattern of autophagy and the crucial roles of V-ATPase in the regulation of different au- tophagy.展开更多
文摘Autophagy is a highly evolutionarily conserved pathway that depends on lysosome to degrade misfolded proteins and damaged organelles. Besides canonical autophagy, studies have shown some chemicals could bypass several core genes to induce autophagy, but the targets and regulatory mechanism is still unclear. In this work one novel chemical, G1, was screened out which could trigger both canonical autophagy and non-canonical autophagy by recruiting Atgl6L1 to pre-autophagosomal site and causing LC3 lipidation. The Gl-induced non-canonical auto- phagy was ULK1, and Beclinl-independent but ubiquitin-like conjugation system-dependent, indicating G1 might target the upstream of Atgl6L1. Moreover, inhibition of V-ATPase by specific V-ATPase inhibitiors could suppress the formation of Gl-indueed autophagosomes in FIP200-defieient MEF cells. While other classic lysosomal inhibi- tors could not block the puneta of Atgl2, Atgl6L1 and LC3, in different stages, suggesting V-ATPase activity in- stead of lysosome function is required for Gl-indueed non-canonical autophagy. These studies broaden the under- standing of different working pattern of autophagy and the crucial roles of V-ATPase in the regulation of different au- tophagy.
